www.nature.com/cddis REVIEW ARTICLE OPEN Protein lysine crotonylation: past, present, perspective ✉ ✉ Gaoyue Jiang1,3, Chunxia Li2,3, Meng Lu2,3, Kefeng Lu 2 and Huihui Li 1 © The Author(s) 2021 Lysine crotonylation has been discovered in histone and non-histone proteins and found to be involved in diverse diseases and biological processes, such as neuropsychiatric disease, carcinogenesis, spermatogenesis, tissue injury, and inflammation. The unique carbon–carbon π-bond structure indicates that lysine crotonylation may use distinct regulatory mechanisms from the widely studied other types of lysine acylation. In this review, we discussed the regulation of lysine crotonylation by enzymatic and non-enzymatic mechanisms, the recognition of substrate proteins, the physiological functions of lysine crotonylation and its cross- talk with other types of modification. The tools and methods for prediction and detection of lysine crotonylation were also described. Cell Death and Disease (2021) 12:703 ; https://doi.org/10.1038/s41419-021-03987-z INTRODUCTION transcriptional activation [10]. Structurally, Kcr is four-carbon in Protein posttranslational modifications (PTMs) are important length and the crotonyl modification contains a carbon–carbon epigenetic regulatory mechanisms involved in diverse biological (C–C) π-bond that results in a unique rigid planar conformation [1]. processes, such as DNA replication, transcription, cell differentia- In this review, we will discuss the enzymatic and non-enzymatic tion, and organismal development. Dysregulation of PTMs is regulation of crotonylation, the cellular and physiological func- associated with a number of diseases, e.g., neuropsychiatric tions of Kcr, the cross-talk between Kcr with other PTMs, and the disease, carcinogenesis, and tissue injury [1]. Due to the prediction tools and detection methods for Kcr. development of high-resolution liquid chromatography with tandem mass spectrometry (LC–MS/MS) for the identification of PTMs, various lysine acylations including acetylation (Kac), REGULATION MECHANISMS OF KCR butyrylation (Kbu), crotonylation (Kcr), propionylation (Kpr), Protein lysine acylation such as Kcr, Ksucc, Kmal, Kglu, and Kbhb malonylation (Kmal), glutarylation (Kglu), benzoylation (Kbz), 2- can be regulated by either enzymatic or non-enzymatic mechan- hydroxyisobutyrylation (Khib), β-hydroxybutyrylation (Kbhb), suc- isms [11]. Both serum and urine have been detected with trace cinylation (Ksucc), and lactylation (Kla) have been identified [2, 3] amounts of short-chain fatty acid (SCFA) crotonate [12, 13]. (Fig. 1). These modifications influence protein structure and Increased crotonate in colon lumen and serum caused elevated modulate their stability, localization, and activity [4]. Based on the histone Kcr [14]. Supplementation with crotonate dramatically chemical properties of lysine modification, acylations are classified enhanced the levels of cellular crotonyl-CoA and histone Kcr [15]. into three groups (Fig. 1): the hydrophobic acyl group, the polar Besides, treatment with crotonate significantly increased global acyl group, and the acidic acyl group [1]. Kcr [16], suggesting the abundance of crotonyl-CoA would be one Crotonylation was initially identified on lysine residues in of the main governing factors of Kcr. histones enriched in the promoter and enhancer regions in both The process converting crotonate into crotonyl-CoA was human somatic and male germinal cells, indicating lysine mediated by Acyl-CoA synthetase short chain family member 2 crotonylation (Kcr) of histone may be an indicator of gene (ACSS2) [15]. Depletion of ACSS2 resulted in drop of cellular expression [5]. The histone Kcr was conserved from yeast to crotonyl-CoA and histone Kcr, indicating crotonate might be the human [5]. Subsequently, non-histone crotonylation was identified endogenous source of crotonyl-CoA [15]. Besides, the SCFA to be particularly enriched in nuclear proteins involved in RNA butyrate through β-oxidation pathway was converted into glu- processing, nucleic acid metabolism, and chromosome organiza- taryl-CoA, and further into crotonyl-CoA by butyryl-CoA dehydro- tion [6]. Later, more studies identified Kcr in non-histone proteins genase (BCDH) [17]. Furthermore, the enzymes that catalyze [7–9]. The crystal structure of the nucleosome containing conversion of butyryl-CoA to crotonyl-CoA during fatty acid crotonylated H3K122cr revealed that H3K122cr did not affect oxidation, acyl-CoA dehydrogenase short chain (ACADS), and acyl- the overall nucleosome structure, but locally impeded the CoA oxidase (ACOX3) were key crotonyl-CoA producers during formation of water-mediated hydrogen bond with DNA backbone, endoderm differentiation [18]. Deletion of ACADS or ACOX3 caused weakened the histone–DNA association, thus favored the drop of intracellular crotonyl-CoA levels without affecting other 1West China Second University Hospital, State Key Laboratory of Biotherapy, and Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, Sichuan University, 610041 Chengdu, China. 2Department of Neurosurgery, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and The Research Units of West ✉ China, Chinese Academy of Medical Sciences, Chengdu, China. 3These authors contributed equally: Gaoyue Jiang, Chunxia Li, Meng Lu. email: [email protected]; [email protected] Edited by B. Zhivotovsky Received: 23 February 2021 Revised: 28 May 2021 Accepted: 31 May 2021 Official journal of CDDpress G. Jiang et al. 2 1234567890();,: Fig. 1 Chemical structures of lysine acylations. Based on their chemical properties, lysine acylations are classified into three groups: the hydrophobic acyl group that extends hydrocarbon chains, including Kac, Kpr, Kbu, Kbz, and Kcr; the polar acyl group includes Kbhb, Khib, and Kla that contain hydroxyl moiety to enable the modified lysine to form hydrogen bonds with other molecules; the acidic acyl group includes Kmal, Ksucc, and Kglu that alter the charge at the lysine residue from +1to–1 at physiological pH [1]. Besides the regulation of Kcr by intracellular crotonyl-CoA levels, several recent studies have demonstrated enzyme-regulation on Kcr. The regulation of Kcr is a dynamic balance between the enzymatic activities of writer and eraser proteins that add and remove modification, respectively. The identification and charac- terization of writers and erasers is essential for classifying the regulatory mechanisms of protein crotonylation (Table 1, Fig. 3). Kcr writers Enzymes that catalyze modification are referred to as writers. However, crotonyl-specific writers have not been identified yet. Previously characterized histone acetyltransferases (HATs) were Fig. 2 The generation of crotonyl-CoA. SCFAs such as crotonate shown to have expanded histone crotonyltransferase (HCT) can be metabolized to crotonyl-CoA by ACCS2 [15]. Besides, SCFA activities. Three major HAT families including p300/CREB-binding butyrate could be converted into butyryl-CoA through β-oxidation protein (p300/CBP), MYST, and GNAT (Gcn5-related N-acetyltras- pathway, and further into crotonyl-CoA by BCDH [17]. ACADS and ferase) were characterized by their sequences and structures ACOX3 that catalyze conversion of butyryl-CoA to crotonyl-CoA (Supplementary Fig. 1), and have been reported as HCTs that use during fatty acid oxidation were demonstrated to be as key crotonyl- crotonyl-CoA as substrate to catalyze Kcr [1]. CoA producers during endoderm differentiation [18]. In amino acid The p300/CBP have both HAT and HCT activities, and p300- metabolism of lysine, hydroxylysine, and tryptophan, GCDH catalyzes catalyzed histone Kcr can directly stimulate transcription [15]. A the oxidation of glutaryl-CoA to crotonyl-CoA and CO2 [19, 20]. CDYL converts crotonyl-CoA into β-hydroxybutyryl-CoA [22]. hydrophobic pocket, predicted to accommodate the aliphatic portion of remodeled acyl-CoA in the active site of p300, was observed in the crystal structures of p300 in complex with propionyl-CoA, crotonyl-CoA, or butyryl-CoA [23]. The size of the pocket and its aliphatic nature restrict against long-chain acyl-CoA acyl-CoAs [18]. During the amino acid metabolism of lysine, variants and instead accommodate short-chain Acyl-CoA such as hydroxylysine and tryptophan, glutaryl-CoA dehydrogenase (GCDH) acetyl-CoA, propionyl-CoA, crotonyl-CoA, or butyryl-CoA without catalyzes the oxidation of glutaryl-CoA to crotonyl-CoA [19, 20]. The major structural rearrangements [23]. However, due to the GCDH deficiency caused accumulation of glutarylcarnitine and restricted size of an aliphatic back pocket and a substrate- neurotoxic glutaric acid, glutaryl-CoA and 3-hydroxyglutaric acid assisted rearrangement of the acyl-CoA chain, the acyltransferase [21]. Furthermore, chromodomain Y-like (CDYL) was reported as a activity of p300 gets weaker with increasing acyl-chain length [23]. crotonyl-CoA hydratase that converts crotonyl-CoA into Still, p300/CBP was considered to be the major HCT in mammalian β-hydroxybutyryl-CoA and negatively regulates histone Kcr [22]. cells [24], the p300/CBP mutants with deficient HAT but Therefore, these studies support the notion that crotonyl-CoA, competent HCT activity substitute the endogenous CBP/p300 to crotonate, and butyrate may drive the occurrence of Kcr (Fig. 2). enhance transcriptional activation [24]. Later, the global Kcr Cell Death and Disease (2021) 12:703 G. Jiang et al. 3 Table 1. writers, erasers and readers of Kcr. Family Targets Enzymes Ref.